A half century ago, Nierenberg and Matthaei discovered the first codon UUU for phenyl alanine using a cell-free translation system from DNase treated E.coli extract. From that time on, the cell-free protein synthesis has been used for the analysis in molecular biology, protein production and protein design, taking advantage of compartment-free experiment. Post-genome proteomics and functional genomics require a high throughput systematic production of proteins. Evolutionary protein engineering requires the translation of a large diversity library. Studies on the translation itself (its molecular mechanism, its origin etc.) require a simplified model system. Cell-free protein synthesis systems are useful for all these themes. This book reviews briefly the history of the translation and the history of its study.

One of the most astonishing molecular events which molecular biology has discovered is the translation process, that is, a Natural digital-to-digital decoding process.

Structural biology found the ribozymatic peptidyl transferase action of ribosome and finally gave us the concept of RNA-makes-Protein. And it suggested the RNA+Protein world was emerged from the RNA world. In the RNA world, the molecular coding process was established probably due to three folds complementarities of RNA molecules as follows: (i) complementary base pairing for amplification, (ii) complementary base pairing for folding and (iii) the complementarity between the surface of the folded RNA and a ligand molecule. The first is related to genotype and the second plus the third are related to phenotype. The phenotype as the molecular function (e.g. specific binding to the ligand) could be digitally encoded in the genotype as the base sequence of the RNA molecule, through the Darwinian selection process just as that of exploiting an RNA aptamer. On the other hand, the decoding process is very simple, i.e., folding and binding. This is the digital-to-real-world decoding. The above mentioned encoding process is also not so complicated, due to the RNA-type genotype-phenotype linking strategy, that is, both on the same molecule. Evolvability of RNA is based on this molecular coding ability. Using this evolvability, evolutionary RNA engineering in these two decades have been creating many kinds of functional artificial RNAs (including new drugs!) and thus indicated the potentiality of RNA molecules and the physico-chemical possibility of the RNA world.

Linking with nucleic acids, polypeptide finally got evolvability and was able to become proteins. The genotype-phenotype linking strategy for Darwinian selection of protein is not so simple. There are three types of the strategy in evolutionary protein engineering as follows: the virus-type, the cell-type and the external intelligence-type.

In the virus–type, mRNA and its protein are bound together just as in the simplest virus particle. In the cell-type, mRNA and its protein are in a same compartment, e.g. a bacterial cell or a micro plate well. In the origin of the translation, what strategy was adopted is not clear. The most complicated aspect of the translation, however, may be the digital-to-digital decoding process. Note: there is no problem in the digital-to-digital encoding because there is no reverse-translation. The encoding process is accomplished via the Darwinian selection using this digital-to-digital decoding and the above mentioned genotype- phenotype linking.

The central issue in the origin of the translation is the establishment of the genetic code table for the digital-to-digital decoding. Present-day standard genetic code table seems to be evolutionally optimized if we admit our twenty amino acids. But whether twenty and these twenty were optimal or not is open question. In fact, protein engineers have been introducing many kinds of non-natural amino acids, tricking the code table for

their purposes. What is the primitive ribosome is also open question. But there is a primitive tRNA model as the first gene. Anyway there should have been a coevolution process of RNA replication and the primitive translation. There are evidences to suggest common unit processes in both RNA replication and the translation. Present-day standard genetic code table is almost universal on the Earth.

And the translational apparatuses are the most conservative molecular machines. These indicate the bottleneck of the biological evolution on the Earth was the establishment of the translation. The enhancement of evolvability of an organism by introducing evolving proteins must overbalance the difficulties of passing the

bottleneck. Thus, protein biosynthesis had two important aspects from the beginning as a matter of course: innovative molecular design and regulated production. These two aspects are also important for modern protein engineers.

The editor of this monograph, Dr. Manish Biyani, is an innovative researcher in the field of cell-free protein synthesis, evolutionary protein engineering and experimental genome analysis. I hope readers enjoy the scope of Dr. Biyani and splendid informative chapters by expert scientists contributed in this book.